Separation of Dihydrofolate Reductase Inhibitors by RP-HPLC and Comparison with Capillary Zone Electrophoresis

The dihydrofolate reductase inhibitors (DHFRIs) are a group of antibiotic compounds with closely related chemical structures. A previous attempt to separate the eight compounds by CZE was successful, but only at low pH (2.1) and high buffer concentration (250mM phosphate). As a result, baseline noise was high. Additionally, baseline resolution was not quite achieved and the separation took 25 minutes. However, the size-based separation indicated that reversed-phase HPLC might be a good alternative. A three-dimensional overlapping resolution mapping (OR_sM) scheme utilised pH, flow rate and percentage organic modifier (for both methanol and acetonitrile). Clear generalised outcomes were observed under isocratic conditions. At higher pHs, where the analytes were largely unionised, retention was excessive. At higher percentages of MeOH or ACN, the more-difficult-to-separate components were poorly resolved. On the other hand, low pHs (2.5 in 50mM phosphate) with low percentages of the organic modifiers (7% ACN or 11% MeOH) but high flow rates (2.3mLmin^–1) yielded better than baseline resolution in 17 minutes. A partially optimised gradient run (at pH 6.5) again yielded far better than baseline resolution in 12 minutes, but required 4 minutes re-equilibration. Hence, the HPLC separations are superior to the CZE separation in all of runtime (40%), resolution and limit of detection (down by 6).